Atherosclerosis is thought to originate from a subtle process of endothelial injury. Vascular endothelium constitutes a non-thrombogenic surface of normally quiescent cells that line blood vessels and regulate molecular and cellular movement across the vessel well. In response to denuding injury, endothelial cells at the wound edge spread and migrate into the vacant area, undergo proliferation and secrete factors that stimulate endothelial and smooth muscle cell growth. These responses provide an important homeostatic mechanism for maintaining normal vascular function. Growth factors such as platelet-derived growth factor (PDGF, which comprises an A chain and/or a B chain) and basic fibroblast growth factor (bFGF) have been implicated to play key roles in the regenerative events following vascular injury. The induction of PDGF gene expression in vascular endothelium may have profound chemotactic and mitogenic effects on the underlying smooth muscle cells and contribute to the structural remodelling that typically occurs in experimental arterial repair, restenosis and in the pathogenesis of atherosclerotic vascular disease (1). Smooth muscle cells are found in both fatty streaks and fibrous atherosclerotic plaques. Their proliferation and ability to form enormous amounts of connective tissue matrix and accumulate lipid are key contributing factors in the development of the atherosclerotic lesion.
Despite a wealth of descriptive studies which correlate the formation of vascular occlusive lesions with the inappropriate expression of these and other growth regulatory molecules (2), a direct link between a transcription factor and the induced expression of a pathophysiologically relevant gene has not yet been demonstrated in the context of arterial injury.
The treatment of occluded coronary arteries currently involves the use of percutaneous transluminal coronary angioplasty (PCTA) or more recently PCTA in conjunction with the placement of a device known as a stent. PCTA is a balloon device that is delivered to the affected site via a catheter and following expansion of the balloon results in physical removal of the blocking plaque or thrombus and enlargement of the local vessel area.
The application of the stent, a fenestrated metallic sleeve, adds additional support to the re-opened vessel and amongst other benefits, prevents the frequency of elastic recoil of the vessel wall. In some of the cases of intervention the benefit of the treatment is short lived and the vessel undergoes reclosure or restenosis. Restenosis is a multi-phased clinical event and can involve elastic recoil in the first instance followed by extensive vascular remodelling and luminal shrinkage. The final stages of the restenotic process involve recruitment and proliferation of smooth muscle cells to create a neo-intimal mass between the elastic lamina and the endothelium. The incidence of restenosis has gradually reduced with the advancement of healthcare methods but is still a significant problem (Kimura et al. (1996) New England Journal of Medicine 335:561-566, Bittl, (1996) New England Journal of Medicine 334:1290-1302).
There is considerable activity focussed on the development of pharmaceuticals to be used as adjuncts to the interventional methods in an attempt to reduce the incidence of restenosis. Some of the classes of drugs under development include:
(a) Anticoagulants--agents such as hirudin and bivalirudin target the formation of thrombin rich clots. PA1 (b) Antiplatelet drugs--suppression of platelet activation can reduce the formation of platelet aggregates and clotting. One approach involves the use of a monoclonal antibody dubbed Abciximab that is specific for the platelet fibrinogen receptor glycoprotein IIb/IIIa. PA1 (c) Antiproliferatives--Trapidil is an antagonist of the PDGF receptor. PDGF is an established stimulator of smooth muscle cell recruitment and proliferation and it is proposed that inhibition of PDGF activity will inhibit this activity. PA1 (d) Antioxidants--compounds such as Probucal are currently under investigation as agents to remove oxidative stress from vessel walls and thus limit the smooth muscle cell proliferation associated with such stress. PA1 (e) Nucleic acid based therapies--antisense and ribozymes directed against specific targets e.g. WO 96/25491, WO 96/20279 and WO 96/11266 PA1 (a) Targeting the Egr-1 gene directly using triple helix (triplex) methods in which a ssDNA molecule can bind to the dsDNA and prevent transcription. PA1 (b) Inhibiting transcription of the Egr-1 gene using nucleic acid transcriptional decoys. Linear sequences can be designed that form a partial intramolecular duplex which encodes a binding site for a defined transcriptional factor. Evidence suggests that Egr-1 transcription is dependent upon the binding of Sp1. AP1 or serum response factors to the promoter region. It could be envisaged that inhibition of this binding of one or more of these transcription factors would inhibit transcription of the Egr-1 gene. PA1 (c) Inhibition of translation of the Egr-1 mRNA using synthetic antisense DNA molecules that do not act as a substrate for RNase H and act by sterically blocking gene expression. PA1 (d) Inhibition of translation of the Egr-1 mRNA by destabilising the mRNA using synthetic antisense DNA molecules that act by directing the RNase H-mediated degradation of the Egr-1 mRNA present in the heteroduplex formed between the antisense DNA and mRNA. PA1 (e) Inhibition of translation of the Egr-1 mRNA by destabilisation of the Egr-1 mRNA by cleavage of the mRNA by sequence-specific hammerhead ribozymes and derivatives of the hammerhead ribozyme such as the Minizymes or Mini-ribozymes or where the ribozyme is derived from: PA1 The composition of the ribozyme could be: PA1 or PA1 The ribozyme could also be either: PA1 (f) Inhibition of translation of the Egr-1 mRNA by cleavage of the mRNA by sequence-specific catalytic molecules composed of DNA. For example molecules described previously by Breaker and Joyce (Breaker and Joyce (1995) Chemistry and Biology 2:655-660) could be developed to cleave Egr-1 mRNA. PA1 (g) Inhibition of Egr-1 activity as a transcription factor using transcriptional decoy methods. A method according to that described in (b) above could be used that would interfere with Egr-1 activity and consequent induction of Egr-1-dependent genes. PA1 (h) Inhibition of the activity of the Egr-1 gene protein by antisense oligonucleotides that have the potential to hybridise specifically to the Egr-1 mRNA and contain four consecutive G residues. These G residues are required for the effect of the oligo in preventing restenosis or atherosclerosis. See WO 96/11266 "Method for inhibiting smooth muscle cell proliferation and oligonucleotides for use therein". PA1 (i) Inhibition of the ability of the Egr-1 gene to bind to its target DNA by drugs that have preference for GC rich sequences. Such drugs include nogalamycin, hedamycin and chromomycin A.sub.3 (Chiang et al J. Biol. Chem. 1996; 271:23999). PA1 (a) Liposomes and liposome-protein conjugates and mixtures. PA1 (b) Using catheters to deliver intra-luminal formulations of the nucleic acid as a solution or in a complex with a liposome. PA1 (c) Catheter delivery to adventitial tissue as a solution or in a complex with a liposome. PA1 (d) Within a polymer such as Pluronic gels or within ethylene vinyl acetate copolymer (EVAc). The polymer will be delivered intra-luminally. PA1 (e) Within a vital-liposome complex, such as Sendal virus. PA1 (f) The nucleic acid may be delivered by a double angioplasty balloon device fixed to catheter. PA1 (g) The nucleic acid could be delivered on a specially prepared stent of the Schatz-Palmaz or derivative type. The stent could be coated with a polymer or agent impregnated with nucleic acid that allows controlled release of the molecules at the vessel wall. PA1 (a) phosphodiester backbone modification by replacement of a non-bridging oxygen atom with sulphur or a methyl group such as in phosphorothioates or methylphosphonates or replacement of phosphodiester backbone with a peptide linked backbone such as in PNAs. PA1 (b) replacement of the 2' hydrogen within the deoxyribose group with a amine, methyl or other alkanes or alkenes or other functional group. PA1 (c) modification of the termini of the oligonucleotide by the addition of an inverted base at the 3' end via 3'--3' linkages. PA1 (d) modification of 5' and 3' by conjugation of other functional groups selected from lipids and steroids such as cholesterol. PA1 (e) phosphodiester backbone modifications in which the phospho-sugar backbone is replaced by a morphilino phophorodiamidate backbone.